Anti ph3 ser10

Immunohistochemistry was performed using formalin‐fixed paraffin‐embedded sections. Following deparaffinization with xylene and rehydration with graded ethanol, antigen retrieval was performed using 0.09% (v/v) unmasking solution (Vector Labs) for 30 min in a steamer. Inactivation of endogenous peroxidases was carried out using 3% hydrogen peroxide (Sigma) for 10 min. Secondary antibody staining and biotin–streptavidin incubation were performed using species‐specific VECTASTAIN Elite ABC Kits (Vector Labs). DAB Peroxidase Substrate Kit (Vector Labs) was utilized for antibody detection. Primary antibodies used were anti‐pH3 Ser10 (1:200, Cell Signalling, 9701), cleaved caspase 3 (1:200, Cell Signalling, 9661) and Phospho‐Met (Tyr1234/1235) (1:300, Cell Signaling, 3077). Tumour sections were visualized under a TissueFAXS slide scanning platform (TissueGnostics, Vienna, Austria). For the pH3 and Casp3, the quantitation was performed using StrataQuest software (TissueGnostics) to determine the percentage of pH3⁺ or Casp3⁺ cells.

Forty-eight hours after transfection with siControl or siLOX, 250,000 cells were plated in glass coverslips in 6-well plates and allowed to attach for 24 hr at 37°C and in a 5% CO₂ atmosphere. Cells were then fixed in 4% paraformaldehyde (PFA) for 15 min. After permeabilization with 70% ethanol, cells were blocked with 5% BSA in PBS-TT (0.5% Tween and 0.1% Triton) for 1 hr at RT. The cells were immunostained for LOX using monoclonal antibody anti-LOX (Abcam), anti-tubulin (Cell Signaling Technology or DSHB Univ. Iowa), and anti-pH3^(Ser10) (Cell Signaling Technology). Then the cells were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 569 for 1 hr (Life Technologies, Thermo Fisher Scientific). DNA was stained with DAPI (Vector Laboratories, Burlingame, CA). For the immunofluorescence staining of the mitotic fractions the samples were fixed in PFA 4%, washed and reconstituted in PBS before overnight incubation on poly-lysine coated cover slips (BD Biosciences) at 4°C. The samples were then stained with Hoechst 33342 (Sigma Aldrich) to detect DNA and with tubulin and LOX antibodies as described above. Fluorescence images were detected by confocal microscope NLO 710 Zeiss, and images were collected using Carl Zeiss Zen Software (Zeiss, Germany).